CA2200563C - Calcium free subtilisin mutants - Google Patents

Abstract

Novel calcium free subtilisin mutants are taught, in particular subtilisins which have been mutated to eliminate amino acids 75-83 and which retain enzymatic activity and stability. Recombinant methods for producing same and recombinant DNA encoding for such subtilisin mutants are also provided.

Description

CALCIUM FREE SUBTILISIN MUTANTS
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grant No. GM42560 awarded by National Institute of Health.
GENERAL OBJECTS OF THE INVENTION
A general object of an aspect of the invention is to provide subtilisin mutants which have been mutated such that they do not bind calcium.
Another object of an aspect of the invention is to provide DNA
sequences which upon expression provide for subtilisin mutants which do not bind calcium.
Another object of an aspect of the invention is to provide subtilisin mutants which comprise specific combinations of mutations which provide for enhanced thermal stability.
Another object of an aspect of the invention is to provide a method for the synthesis of a subtilisin mutant which does not bind calcium by the expression of a subtilisin DNA which comprises one or more substitution, deletion or addition mutations in a suitable recombinant host cell.
A more specific object of an aspect of the invention is to provide class I
subtilise mutants, in particular BPN' mutants which have been mutated such that they do not bind calcium.
Another specific object of an aspect of the invention is to provide DNA
sequences which upon expression result in class I subtilise mutants, and in particular BPN' mutants which do not bind calcium.
Another specific object of an aspect of the invention is to provide a method for making subtilisin I-S1 or I-S2 mutants, and in particular BPN' mutants which do not bind calcium by expression of a class I subtilise mutant DNA sequence, and more specifically a BPN' DNA coding sequence which comprises one or more substitution, addition or deletion mutations in a suitable recombinant host cell.

Yet another specific object of an aspect of the invention is to provide mutant subtilisin I-S1 or I-S2, and more specifically BPN' mutants which do not bind calcium and which further comprise particular combinations of mutations which provide for enhanced thermal stability, or which restore cooperativity to the folding reaction.
The subtilisin mutants of the present invention are to be utilized in applications where subtilisins find current usage. Given that these mutants do not bind calcium they should be particularly well suited for use in industrial environments which comprise chelating agents, e.g, detergent compositions, which substantially reduces the activity of wild-type calcium binding subtilisins.
BACKGROUND OF THE INVENTION
(1 ) Field of the Invention The present invention relates to subtilisin proteins which have been modified to eliminate calcium binding. More particularly, the present invention relates to novel subtilisin I-S1 and I-S2 subtilisin mutants, specifically BPN' mutants wherein the calcium A-binding loop has been deleted, specifically wherein amino acids 75-83 have been deleted, and which may additionally comprise one or more other mutations, e.g., subtilisin modifications, which provide for enhanced thermal stability and/or mutations which restore cooperativity to the folding reaction.
(2) Description of the Related Art Subtilisin is an unusual example of a monomeric protein with a substantial kinetic barrier to folding and unfolding. A well known example thereof, subtilisin BPN' is a 275 amino acid serine protease secreted by Bacillus amyloliquefaciens. This enzyme is of considerable industrial importance and has been the subject of numerous protein engineering studies (Siezen et al., Protein Enaineerina 4:719-737 (1991 ); Bryan, Pharmaceutical Biotechnoloav 3(8):147-181 (1992); Wells et al., Trends Biochem. Sci.
13:291-297 (1988)). The amino acid sequence for subtilisin BPN' is known in the art and may be found in Vasantha et al., J. Bacteriol. 159:811-819 (1984). The amino acid sequence as found therein is provided herein and referred to as SEQUENCE ID N0:1.
Throughout the application, when Applicants refer to the amino acid sequence of subtilisin BPN' or its mutants, they are referring to the amino acid sequence as listed therein.
Subtilisin is a serine protease produced by Gram positive bacteria or by fungi. The amino acid sequences of numerous subtilisins are known. (Siezen et al., Protein Engineering 4:719-737 (1991 )). These include five subtilisins from Bacillus strains, for example, subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus, and mesenticopeptidase.
(Vasantha et al., "Gene for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open-reading frame between the regions coding for signal sequence and mature protein," J. Bacteriol.
159:811-819 (1984); Jacobs et al., "Cloning sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis," Nucleic Acids Res. 13:8913-8926 (1985); Nedkov et al., "Determination of the complete amino acid sequence of subtilisin DY and its comparison with the primary structure of the subtilisin BPN', Carlsberg and amylosacchariticus," Biol. Chem. Hoppe-Seyler 366:421-430 (9185); Kurihara et al., "Subtilisin amylosacchariticus," J. Biol.
Chem. 247:5619-5631 (1972); and Svendsen et al., "Complete amino acid sequence of alkaline mesentericopeptidase," FEBS Lett. 196:228-232 (1986)).
The amino acid sequences of subtilisins from two fungal proteases are known: proteinase K from Tritirachium albam (Jany et al., "Proteinase K from Tritirachium albam Limber," Biol. Chem. Hoppe-Seyler 366:485-492(1985)) ?5 and thermomycolase from the thermophilic fungus, Malbranchea pulchella (Gaucher et al., "Endopeptidases: Thermomycolin," Methods Enzymol.
45:415-433 (1976)).
These enzymes have been shown to be related to subtilisin BPN', not only through their primary sequences and enzymological properties, but also by comparison of x-ray crystallographic data. (McPhalen et al., "Crystal and r w0 96109396 ~ fi ~ pCTlUS951055Z0 molecular structure of the inhibitor eglin from leeches in complex with subtilisin Carlsberg," S L.ett., 188:55-58 (1985) and Pahler et al., "Three-dimensional structure of fungal proteinase K reveals similarity to bacterial subtiIisin,"
MBO
~, 3:1311-1314 (1984)).
SubtiIisin BPN' is an example of a particular subtiIisin gene secreted by Bacillur amyloliquefaciens. This gene has been cloned, sequenced and expressed at high levels from its natural promoter sequences in Bacillus subtilis. The subtiIisin BPN' structure has been highly refined (R = 0.14) to 1.3 A
resolution and has revealed structural details for two ion binding sites (Finzel et al. , J. Cell.
IO Biochem. SuppI 10A:272 (1986); PantoIiano et al., Biochemistry 27:8311-8317 (1988); McPhaIen et al., Biochemistry 27: 6582-6598 (1988)). One of these (sire A) binds Ca2+ with high affinity and is located near the N-terminus, while the other (site B) binds calcium and other cations much more weakly and is located about 32 A away (Figure I). Structural evidence for two calcium binding sites IS was also reported by Bode et al., Bur. J. Biochem 166:673-692 (1987) for the homologous enzyme, subtiIisin Carlsberg.
Further in this regard, the primary calcium binding site in all of the subtiIisins in groups I-SI and I-S2 (Siezen et al., 1991, Table 7) are formed from almost identical nine residue loops in the identical position of helix C. X-ray . 20 structures of the I-S 1 subtilisins BPN' and Carlsberg, as well as the I-subtiIisin Savinase, have been determined to high resolution. A comparison of these structures demonstrates that all three have~~almost identical calcium A-sites.
The x-ray structure of the class I subtilase, thermitase from Thermoacrinomyces vulgaris, is also !mown. Though the overall homology of 25 BPN' to thermitase is much lower than the homology of BPN' to I-S 1 and I-subtiIisins, thermitase has been shown to have an analogous calcium A-site. In the case of thermitase, the loop is a~ ten residue interruption at the identical site in helix C.
Calcium binding sites are common features of extracellular microbial 30 proteases probably because of their Large contribution to both thermodynamic and kinetic stability (Matthews et al., J. Biol. Chem. 249:8030-8044 (1974);
Voordouw et al., Biochemistry 15:3716-3724 (1976); Betzel et al., Protein Engineering 3:161-172 (1990); Gros et al., J. Biol. Chem. 266:2953-2961 (1991 )). The thermodynamic and kinetic stability of subtilisin is believed to be 5 necessitated by the rigors of the extracellular environment into which subtilisin is secreted, which by virtue of its own presence is protease-filled.
Accordingly, high activation barriers to unfolding may be essential to lock the native conformation and prevent transient unfolding and proteolysis.
Unfortunately, the major industrial uses of subtilisins are in environments containing high concentrations of metal chelators, which strip calcium from subtilisin and compromise its stability. It would, therefore, be of great practical significance to create a highly stable subtilisin which is independent of calcium.
The present inventors have previously used several strategies to increase the stability of subtilisin to thermal denaturation by assuming simple thermodynamic models to approximate the unfolding transition (Pantoliano et al., Biochemistry 26:2077-2082 (1987); Pantoliano et al., Biochemistry 27:8311-8317 (1988); Pantoliano et al., Biochemistry 28:7205-7213 (1989);
Rollence et al., CRC Crit. Rev. Biotechnol. 8:217-224 (1988). However, improved subtilisin mutants which are stable in industrial environments, e.g., which comprise metal chelators, and which do not bind calcium, are currently not available.
SUMMARY OF THE INVENTION
Accordingly, it is an object of an aspect of the invention to provide mutated or modified subtilisin enzymes, e.g., class I subtilises, which have been modified to eliminate calcium binding. As used in this invention, the term "mutated or modified subtilisin" is meant to include any serine protease enzyme which has been modified to eliminate calcium binding. This includes, in particular, subtilisin BFN' and serine proteases which are homologous to subtilisin BPN', in particular class I subtilises. However, as used herein, and under the definition of mutated or modified subtilisin enzyme, the mutations of this invention may be w0 96!09396 PCTlUS95/05520 22 00 ~fi3 introduced into any serine protease which has at least 50 % , and preferably amino acid sequence identity with the sequences referenced above for subtilisin BPN', subtilisin Carlsberg, subtilisin DY, subtilisin amylosacchariticus, mesenticopeptidase, thermitase, or Savinase and, therefore, may be considered homologous.
The mutated subtilisin enzymes of this invention are more stable in the presence of metal chelators and may also comprise enhanced thermal stability in comparison to native or wild-type subtilisin. Thermal stability is a good indicator of the overall robustness of a protein. Proteins of high thermal stability often are stable in the presence of chaotropic agents, detergents, and under other conditions, which normally tend to inactivate proteins. Thermally stable proteins are, therefore, expected to be useful for many industrial and therapeutic applications in which resistance to high temperature, harsh solvent conditions or extended shelf life is required.
. It has been further discovered that combining individual stabilizing mutations in subtilisin frequently results in approximately additive increases in the free energy of stabilization. Thermodynamic stability has also been shown to be related to resistance to irreversible inactivation at high temperature and high pH. The single-site changes of this invention individually do not exceed a 1.5 Kcal/moI contribution to the free energy of folding. However, these small incremental increases in the free energy of stabilization result in dramatic increases in overall stability when mutations are combined, since the total free energy of folding for most proteins is in the range of 5-15 Kcals/mol (Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman and Company, New York (1984)).
X-ray crystallographic analysis of several combination mutants reveals that conformational changes associated with each mutation tend to be highly localized with minimal distortion of the backbone structure. Thus, very large increases in stability can be achieved with no radical changes in the tertiary protein structure and only minor independent alterations in the amino acid ~2 00 5~3 sequence. As previously suggested (Holmes et al, J. Mol. Blot. 160:623 (1982)), contributions to the free energy of stabilization can be gained in different ways, including improved hydrogen bonding and hydrophobic interactions in the folded form and decreased chain entropy of the unfolded enzyme. 'This is significant since thetmostable enzymes generally have more extended half lives at broader temperature ranges, thereby improving bio-reactor and shelf Iife performance.
As noted supra, the invention provides subtilisin mutants which comprise one or more deletion, substitution or addition mutations which provide for the IO elimination of calcium binding. Preferably, this will be effected by deletion, substitution or insertion of amino acids into the calcium A-site, which in the case of class I subtilases comprises 9 amino acid residues in helix C. In the case of subtilisin BPN', the subtilisin mutants will preferably comprise one or more addition, deletion or substitution mutations of the amino acids at positions 75-83, and most preferably will comprise the deletion of amino acids 75-83, of SEQUENCE ID NO:I. The deletion of amino acids 75-83 has been discovered to effectively eliminate calcium binding to the resultant subtilisin mutant while still providing for subtilisin BPN' proteins having enzymatic activity.
Such subtilisin mutants lacking amino acids 75-83 of SEQUENCE ID
NO:1 may further include one or more additional amino acid mutations in the sequence, e.g., mutations which provide for reduced proteolysis. It is another object of the invention to produce subtilisin mutants lacking calcium binding activity which have been further mutated to restore cooperativity to the folding reaction and thereby enhance proteolytic stability. It is another object of the invention to provide thermostable subtilisin mutants which further do not bind calcium and comprise specifc combinations of mutations which provide for substantially enhanced thermal stability.
In particular, the subtiiisin mutants of the present invention will include subtilisins from Bacillus strains, such as subtilisin BPN', subtilisin Carlsberg, _$_ subtilisin DY, subtilisin amylosacchariticus and subtilisin mesenticopeptidase, which comprise one or more deletion, substitution or addition mutations.
The present invention further provides for subtilisin mutants lacking amino acids 75-83 of SEQUENCE ID N0:1, which have new protein-protein interactions engineered in the regions around the deletion leading to large improvements in stability. More specifically, mutations at ten specific sites in subtilisin BPN' and its homologues are provided, seven of which individually, and in combination, have been found to increase the stability of the subtilisin protein. Improved calcium-free subtilisins are thus provided by the present invention.
In accordance with one embodiment, the present invention provides an enzymatically active subtilisin protein which has been mutated to delete amino acids at positions 75-83 of mature subtilisin BPN' to eliminate the ability of the subtilisin protein to bind calcium at a high affinity calcium binding site, wherein the mutated subtilisin protein further includes at least one additional deletion, substitution or addition mutation which enhances the stability of the mutant protein in at least one region which corresponds to a region of the mature subtilisin BPN' selected from the group of mature subtilisin BPN' regions consisting of the N-terminal acids 1-8, the w-loop amino acids 36-45 and the a-helix amino acids 70-74.
In accordance with a further embodiment, the present invention provides a recombinant method which provides for the expression of an enzymatically active subtilisin protein which has been mutated to delete amino acids at positions 75-83 of mature subtilisin BPN' to eliminate the ability of the subtilisin protein to bind calcium at a high affinity calcium binding site, wherein the mutated subtilisin protein further comprises at least one deletion, substitution or addition mutation which enhances the stability of the mutant protein in at least one region which corresponds to a region of the mature subtilisin BPN' selected from the group of mature subtilisin BPN' regions consisting of the N-terminal amino acids 1-8, the w-loop amino acids 36-45 and the a-helix amino acids 70-74, the method comprising:
(a) transforming a recombinant host cell with an expression vector which contains a DNA which encodes the mutated enzymatically active subtilisin protein;

-8a-(b) culturing the host cell under conditions which provide the expression of the mutated enzymatically active subtilisin; and (c) recovering the expressed mutated enzymatically active subtilisin from the microbial host.
In accordance with a further embodiment, the present invention provides a recombinant DNA which encodes a subtilisin protein which has been mutated to delete amino acids at positions 75-83 of mature subtilisin BPN' to eliminate the ability of the subtilisin protein to bind calcium at a high affinity calcium binding site, wherein the mutated subtilisin protein further comprises one or more deletion, substitution or addition mutation which enhances the stability of the mutant protein in at least one region which corresponds to a region of the mature subtilisin BPN' selected from the group of mature subtilisin BPN' regions consisting of the N-terminal amino acids 1-8, the c~-loop amino acids 36-45 and the a-helix amino acids 70-74 and which mutated subtilisin protein retains enzymatic activity and stability.
In accordance with a further embodiment, the present invention provides an enzymatically active subtilisin protein which has been mutated to delete amino acids at positions 75-83 of mature subtilisin BPN' to eliminate the ability of the subtilisin protein to bind calcium at a high affinity calcium binding site, wherein the mutated subtilisin protein includes at least one additional deletion, substitution or addition mutation which enhances the stability of the mutant protein in at least one region which corresponds to a region of the mature subtilisin BPN' selected from the group of mature subtilisin BPN' regions consisting of the N-terminal acids 1-8, the w-loop amino acids 36-45, the a-helix amino acids 70-74 and the helix turn amino acids 84-89, and wherein a mutation in the N-terminal amino acids 1-8, the c~-loop amino acids 36-45, the a-helix amino acids 70-74 and the helix turn amino acids 84-89 is free of disulfide cross-links.
In accordance with a further embodiment, the present invention provides a recombinant method which provides for the expression of an enzymatically active subtilisin protein which has been mutated to delete amino acids at positions 75-83 of mature subtilisin BPN' to eliminate the ability of the subtilisin protein to bind calcium at a high affinity calcium binding site, wherein -8b-the mutated subtilisin protein and includes at least one deletion, substitution or addition mutation which enhances the stability of the mutant protein in at least one region which corresponds to a region of the mature subtilisin BPN' selected from the group of mature subtilisin BPN' regions consisting of the N-terminal amino acids 1-8, the w-loop amino acids 36-45, the a-helix amino acids 70-74 and the helix turn amino acids 84-89, and wherein a mutation in the N-terminal amino acids 1-8, the w-loop amino acids 36-45, the a-helix amino acids 70-74 and the helix turn amino acids 84-89 is free of disulfide cross-links, the method comprising:
(a) transforming a recombinant host cell with an expression vector which contains a DNA which encodes the mutated enzymatically active subtilisin protein;
(b) culturing the host cell under conditions which provide the expression of the mutated enzymatically active subtilisin; and (c) recovering the expressed mutated enzymatically active subtilisin from the microbial host.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 7. X-ray Crystal structure of S15 subtilisin A. a-carbon plot shows the positions of mutations as noted. The numbering of wild type subtilisin is kept. Dotted spheres show the position of calcium at the weak ion binding site (B-site) and the former position of the high affinity binding site (A-site). The A-site loop (dashed line) is absent in this mutant. N- and C- termini are indicated. The N-terminus is disordered (dotted line).
B. Close-up view of the A-site deletion. The loop from S12 subtilisin is shown as a dotted line with the continuous helix of S15. Superimposed is the 3* sigma difference electron density (F012-F015, phases from S15) showing the deleted A-site loop.
Figure 2. X-ray crystal structure of the calcium A-site region of S12 subtilisin. Calcium is shown as a dotted sphere with one-half the van der Waals radius. Dashed lines are coordination bonds, while dotted lines represent hydrogen bonds under 3.2 A.

i WO 9bl09396 PCT/US95I05520 Figure 3. Differential Scanning Calorimetry. The calorimetric scans of apo-S 12 (Tm = 63 . S ° C) and S 15 (T~ = 63 . 0 ° C) are shown.
Measurements were performed with a Hart 7707 DSC (differential scanning calorimetry) heat conduction scanning microcalorimeter as described (Pantoliano et al., Biochemistry 28:7205-7213 (I989)). Sample conditions were 50 mM of glycine, a pH of 9.63, a scan rate of 0.5°C/min. Excess heat capacity is measured in units of fcJl°. The calorimeter ampoules contained I.78 mg of protein.
Figure 4. Titration calorimetry of subtilisin S I 1. The heat of calcium binding for successive additions of calcium are plotted vs. the ratio of [Ca]/(P].
The data are best fit by a calculated binding curve assuming a binding constant of 7 x 106 and OH equal to I1.3 kcaI/moI using equation (I) from the text. For comparison, calculated curves assuming Ka = 1 x 106 and I x 10g are also shown. In this titration, [PJ = 100EcM and the temperature was 25°C.
Figure S. Kinetics of calcium dissociation from subtilisin S 1 I as a IS function of temperature. I~cM subtilisin SI1 was added to 10~,M Quint at ' time = 0. Calcium dissociates from subtilisin and binds to Quint until a new equilibrium is achieved. The rate of calcium dissociation is followed by the increase in fluorescence of Quint when it binds to calcium.
A. The log of the percent of the protein bound to calcium is plotted vs. time. The kinetics of dissociation at four temperatures are shown. The dissociation follows first order kinetics for the first 25 % of the reaction.
As this is weal before equilibrium is approached, reassociation of calcium can be neglected.
B. Temperature dependence of the rate of calcium dissociation from S 15 subtilisin in the presence of excess Quint, pH 7.4 and over a temperature range of 25-45°C. The natural Iog of the equilibrium constant for the transition state (calculated from the Eyring equation) is plotted vs. the reciprocal of the t WO 96!09396 PCT/US95105520 2~ 00 563 absolute temperature. The Line is f t according to equation (3) in the text with To = 298 K.
Figure 6. Analysis of subtilisin refolding monitored by circular dichroism (CD).
6 A. CD spectra are shown for S15 as follows: (I) S15 in 25 mM
HgP04 at pH 1.85; (2) SIS denatured at pH I.85 and then neutralized to pH 7.5 by the addition of NaOH; (3) S15 denatured at pH 1.85 and neutralized to pH
7.5, 30 minutes after the addition of KCI to 0.6 M; and (4) Native SIS
subtilisin.
Protein concentrations of all samples was I ~cM.
B. Kinetics of refolding of S 15. Samples were denatured at pH 1.85 and then the pH was adjusted to 7.5. At time 0, KCl was added to the denatured protein. Recovery of native structure was followed at 222 nm at KCl concentrations of 0.3 M and 0.6 M. The 0.6 M sample after 30 minutes of refolding was then used to record the corresponding spectrum in part A.
~ Figure 7. Kinetics of refolding of S IS as a function of ionic strength.
A. The Iog of the percent unfolded protein is plotted vs. time. The kinetics of refolding are shown at four ionic strengths. The amount of refolding was determined by circular dichroism (CD) from the increase in negative ellipticiry at 222 nm. I00 % folding is determined from the signal at 222 am for native S 15 at the same concentration and 0 % folding is determined from the signal for acid-denatured SIS. The refolding approximately follows first order kinetics for the first 90% of the reaction. Refolding was carried out at 25°C.
B. The Iog of first order rate constants for refolding obtained by CD
or fluorescence measurements at 25°C were plotted as a function of log of ionic - strength. Ionic strength was varied from I=0.25 to I=I.S. The rate of ' refolding increases linearly with log I. A ten-fold increase in I results in an approximately 90-fold increase in the refolding rate.

WO 96109396 2 2 0 0 5 6 3 PGTlUS95/055Z0 -I I-Figure 8. Temperature dependence of the refolding rate of SIS
subtilisin in 0.6 M KCI, 23 nM KP04 pH 7.3. The natural log of the equilibrium constant for the transition state (calculated from the Eyring equation) is plotted vs. the reciprocal of the absolute temperature. The Line is f t according to equation 3 in the text with To=298 K.
Figure 9. X-ray crystal structure of the weak ion binding region of S 15 subtilisin. Coordination bonds are shown as dashed Lines. Note the preponderance of charged amino acids.
DESCRIPTION OF THE PREFERRED EMBODI11~IENTS
IO As discussed supra, calcium binding contributes substantially to the thermo-dynamic and kinetic-stability of extracellular microbial proteases.
Moreover, with respect to subtilisin, high activation barriers to unfolding may be essential to retain the native conformation and to prevent transient unfolding and proteolysis given the protease-filled environment where subtilisin is secreted and as a result of auto-degradation. The unfolding reaction of subtilisin can be divided into two parts as follows:
N(Ca) a N ~r U
where N(Ca) is the native form of subtilisin with calcium bound to the high affinity calcium-binding site A (Finzel et al., J. Cell. Biochem. Snppl.
10A:272 (1986); Pantoliano et al., Biochemistry 27:8311-8317 (1988); McEhalen et aL, Biochemistry 27:6582-6598 (1988)); N is the folded protein without calcium bound; and U is the unfolded protein. Subtilisin is a relatively stable protein whose stability is in Large part mediated by the high affinity calcium site (Voordouw et aL, Biochemistry 15:3716-3724 (1976); Pantoliano et al., Eiochemistrv 27:8311-8317 (1988)). The melting temperature of subtilisin at pH

8.0 in the presence of ~cmolar concentrations of calcium is approximately 75°C
and approximately 56°C in the presence of excess EDTA (Takehashi et al., biochemistry 20:6185-6190 (1981); Bryan et al., Proc. Natl. Acad. Sci. USA, t 4 WO 96!09396 ~ ~ ~ ~ ~ ~ ~ PCTlUS95105520 83:3743-3745 (1986b)). Previous calorimetric studies of the calcium-free (apoenzyme, i.e., protein portion of enzyme) form of subtilisin indicated that it is of marginal stability at 25°C with a DG of unfolding of <5 kcal/moI
(Pantoliano et al., Biochemistry 28:7205-7213 (1989)). Because calcium is such an integral part of the subtilisin structure, the apoenzyme is thought to be a folding intermediate of subtilisin.
In order to independently examine the two phases of the folding process, the present inventors constructed a series of mutant subtilisins. First, all proteolytic activity was eliminated in order to prevent auto-degradation from occurring during the unfolding and refolding reactions. This may be accomplished, for example, by converting the active-site serine 221 to cysteine.l This mutation has little effect oa the thermal denaturation temperature of subtilisin, but reduces peptidase activity of subtilisin by a factor of approximately 3 x 104 (Abrahmsen et al., Biochemistry 30:4151-4159 (1991)). This mutant, therefore, allows the folding of subtilisin to be studied without the complications of proteolysis. In the present specifcation, a shorthand for denoting amino acid substitutions employs the single letter amino acid code of the amino acid to be substituted, followed by the number designating where in the amino acid seduence the substitution will be made, and followed by the single letter code of the amino acid to be inserted therein. For example, S221C denotes the substitution of serine 221 to cysteine. The subtilisin mutant with this single amino acid substitution is denoted subtilisin S221C. The resulting S221C
subtilisin mutant is designated S 1.
The subtilisin may be further mutated in order to make the relatively unstable apoenzyme easier to produce and purify. Versions of S I with three or four additional mutations, for example, MSOF, Q206I, Y217K and N218S, may also be employed in the method of the present invention. Such further mutations 1 ~ The S221A mutant was originally constructed for this propose. The mature form of this mutant was heterogeneous on its N-terminus, however, presumably due to some incorrect processing of the pro-enzyme.

WO 96/09396 ~ 6 ~ PCTIUS95I05520 cumulatively increase the free energy of unfolding by 3.0 kcal/mol and increase the thermal denaturation temperature of the apoenzyme by I1.5 ° C
(Pantoliano et al., Biochemistry 28:7205-7213 (1989)). The mutant containing the MSOF, Q206I, Y2I7K, N218S and S22IC mutations is designated SII and the mutant containing the M50F, Y217K, N2I8S and S221C is designated SI2.2 In order to produce a subtilisin BPN' protein lacidng calcium binding activity, the present inventors elected to delete the binding loop in the calcium A-site to engineer a novel calcium-free subtilisin protein. This Ioop comprises an interruption in the subtilisin BPN' a-helix involving amino acids 63-85 of SEQITENCE m NO:I (McPhalen and James 1988). Residues 75-83 of the subtilisin BPN' protein form a Ioop which interrupts the last turn of the 14-residue alpha helix involving amino acids 63-85 [SEQUENCE m NO:1].3 Four of the carbonyl oxygen ligands to the calcium are provided by a Ioop composed of amino acids 75-83 [SEQUENCE m NO: I]. The geometry of the ligands is that of a pentagonal bipyramid whose axis runs through the carbonyls of amino acids 75 and 79. On one side of the loop is the bidentate carboxylate (D41), while on the other side is the N-terminus of the protein and the side chain of Q2.
The seven coordination distances range from 2.3 to 2.6 A, the shortest being to the aspartyl carbozylate. Three hydrogen bonds link the N terminal segment to Ioop residues 78-82 in parallel-beta arrangement. A high affinity calcium binding site is a common feature of subtilisins which make large contributions to their high stability. By binding at specific sites in the subtilisin tertiary structure, The specific activities of S 1 I, S 12 and S 15 against the synthetic substrate, SAAPFna, are the same. (S.A. = 0.0024 U/mg at 25°C, pH 8.0). These measurements were performed on protein freshly purified on a mercury affinity column.
This set of nine residues was chosen for deletion, as opposed to 74-82 (those actually belonging to the loop) out of preference for AIa 74 rather than Gly 83 in the resulting continuous helix. Alanine has a higher statistical likelihood for occurrence in «-helix, due to glycine's broader range of accessible backbone conformations.

s 2~ p0 X63 WO 9b/09396 PGT/US95/055Z0 ternary structure, calcium contributes its binding free energy to the stability of the native state. In the present invention, site-directed mutagenesis was used to delete amino acids 75-83 of SEQUENCE 1D NO:1, which creates an uninterrupted helix and abolishes the calcium binding potential at site A
(Figure lA and 1B).
The present inventors believed that a stabilization strategy based around calcium binding would allow survival in the eztracellular environment. Since the major industrial uses of subtiIisins are in environments containing high concentrations of metal chelators, it was of great practical significance for the present inventors to produce a stable subtilisin which is independent of calcium and, therefore, unaffected by the presence of metal cheIating agents.
While the present inventors chose to eliminate calcium binding by the removal of these amino acids, it should be possible to eliminate calcium binding by other mutations, e.g., substitution of one or more of the amino acids at positions 75-83 with alternative amino acids and by insertion, substitution and/or deletion of amino acids proximate to positions 75-83. This may also be accomplished by site-specific mutagenesis.
Additionally, because this Ioop is a common feature of subtilisins, it is expected that equivalent mutations for other subtiIisins, in particular class I
subtilises, e.g., by site-specific mutagenesis, will likewise eliminate calcium binding and provide for enzymaticaIly active mutants.
In particular, the present inventors synthesized by site-specific mutagenesis three subtilisin BPN' DNA's which have been mutated to eliminate amino acids 75-83 involved in calcium binding and which further comprise additional substitution mutations. These mutated subtilisin BPN' DNA's, upon expression of the DNA, provide for subtilisin proteins having enhanced thermal stabiIiry and/or which are resistant to proteolysis.
The specific subtilisin BPN' mutants synthesized by the present inventors are designated in this application as S15, 539, 546, S47, S68, 573, S79, and S86. The specific point mutations set forth in the present application identify the WO 96/09396 PC'TlUS95/05520 P~~lat' amino acids in the subtiIisin BpN~ amino acid sequence, as set forth in SEQUENCE ID NO:I, that are mutated in accordance with the present invention. For example, the SIS mutant comprises a deletion of amino acids 75-83 and additionally comprises the following substitution mutations: S221C, N218S, MSOF and Y217K. The S39 mutant similarly comprises a deletion of amino acids 75-83 and additionally comprises the following substitution mutations: 5221 C, PSA, N218S, MSOF and Y2I7K. The S4b mutant comprises a deletion of amino acids 75-83 and additionally comprises the following substitution mutations: MSOF, Y2I7K and N218S. The S47 mutant similarly IO comprises a deletion of amino acids 75-83 and additionally comprises the following substitution mutations: PSA, N218S, MSOF and YZI7K. The S68 mutant comprises a deletion of amino acids 75-83 and additionally comprises the following substitution mutations: PSS, N2i8S, MSOF,and Y2I7K. The S73 mutant comprises a deletion of amino acids 75-83 as well as the following substitution mutations: Q2K, MSOF, A73L, Q206V, Y217K and N218S. The S79 mutant comprises a deletion of amino, acids 75-83 and additional comprises the following substitution mutations: Q2K, MSOF, A73L, Q206C, Y217K and N218S. Finally, the S86 mutant comprises a deletion of amino acids 75-83 as well as the following substitution mutations: Q2K, S3C, MSOF, A73L, Q206C, Y217K and N2I8S. The specific activities of the proteoIytically active S46, S47, S68, 573, S79 and S86 subtilisins have been found to be similar or enhanced in relation to the wild-type enzyme.
The various 075-83 subtilisins which were synthesized by the inventors are shown in Table I, below. The particular points of mutation in the amino acid sequence of subtiIisin BPN~ amino acid sequence, as set forth in SEQUENCE ID
NO:I, are identified. The synthesis of these mutants is described in more detail in, fi-a.

22 00 ~s3 WO 96!09396 ,~ ~ PCTlUS95105520 _o .
a _c a a~
'Q
C
a :a > rA
.G R
C
O
eQ
'a N_ C
H v C.
'ZJ V
4~
V
'3 C
V
N a a R .C
R
O
C
_O
C V
E _' H

U
U
R
c E
H
_.r.
~R ~_>
G
U
c 3 H Q\
R M
R 'C
C
ea O ..~
N Gn C .N..
~0 N _ Cr wU 96/09396 PGT/US95/05520 _;,_ In order to understand the contribution of calcium binding to the stability of subtilisin, the thermodynamics and kinetics of calcium binding to the high affinity calcium A-site were measured by microcalorimetry and fluorescence spectroscopy. Calcium binding is an enthalpically driven process with an association constant (Ka) equal to 7 x 106 M-1. The kinetic barrier to calcium removal from the A-site (23 kcaIlmol) is substantially larger than the standard free energy of binding (9.3 kcaI/mol). The kinetics of calcium dissociation from subtilisin (e.g., in excess EDTA) are accordingly very slow. For example, the half life (tlJ2) of calcium dissociation from subtilisin, i.e., the time for half of IO the calcium to dissociate from subtilisin, is I.3 hours at 25°C.
X-ray crystallography shows that except for the region of the deleted calcium-binding loop, the structure of the subtilisin mutants and the wild type protein are very similar. The N-terminus of the wild-type protein lies beside the site A loop and furnishes one calcium coordination ligand, the side chain oxygen of Q2. In X75-83 subtilisin, the loop is gone, leaving residues 1-4.
disordered.
These first four residues are disordered in the X-ray structure since all its interactions were with the calcium loop. N-terminal sequencing confirms that the first four amino acids are present, confirming that processing occurs at the normal site. The helix is shown to be uninterrupted and shows normal helical geometry over its entire length. X-ray crystallography further shows that the structures of subtilisin with and without the deletion superimpose with a root mean square (r.m.s.) difference between 26I a-carbon positions of 0.17 A, and are remarkably similar considering the size of the deletion. Diffuse difference density and higher temperature factors, however, indicate some disorder in the newly exposed residues adjacent to the deletion.
While the elimination of calcium binding is advantageous since it produces proteins that are more stable in the presence of metal cheiators, it is disadvantageous in at least one respect. Specifically, the elimination of the calcium loop without any other compensating mutations results in the destabilization of the native state relative to the partially folded states and, WO 96!09396 PCT/US95/055Z0 22 00 56~
-18_ therefore, a loss of cooperativity in folding. The present inventors thus sought to further genetically engineer the subtilisin S 15 BPN' protein lacking amino acids 75-83 in order to restore cooperativiry to the folding reaction. In most well designed proteins all pare of the molecule are interdependent, making the unfolding reaction highly cooperative. Cooperativiry of the folding reaction allows proteins to achieve sufficient stabilities of the native state for proper function since the overall stability of the native conformation is roughly the sum of all local interactions.
Therefore, while the 075-83 subtilisin is an example of an engineered subtilisin which is active and stable in the absence of calcium, the present inventors sought to improve this protein by further mutation. The design of a particular' highly stable calcium-free subtilisin relies on an iterative engineering cycle. The present inventors found that the requisite first step in the cycle was to greatly diminish the proteokytic activity of subtilisin. This is necessary because calcium contributes greatly to the conformational stability of subtilisin and the early versions of calcium-free subtilisin are susceptible to proteokysis.
After reducing the susceptibility to proteokysis, the next step in the cycle was to eliminate sequences essential for calcium binding, i.e., the A-site. Although the S 15 075-83 subtilisin is much tress stable than the wild type subtilisin in the presence of calcium, this mutant is more stable than wild type subtilisin in the presence of the metal chekator F.DTA.
Accordingly, the third step was to improve the stability of the calcium-free subtiIisin protein. To improve the stability of calcium-free subtilisin, the present inventors next tried to create a home for the disordered N-terminal residues. In order to create a highly stable calcium-free subtilisin, the N-terminal part of the protein which is destabilized by the deletion of the calcium A-loop may be modified. For example, the N-terminus which is disordered may be deleted or extended. This, however, is problematic because the requirements for processing the propeptide from the mature protein are not known. It is known, however, that the processing site is not determined by amino acid WO 96!09396 2 2 D 4 ~ 6 3 p~/Ug95105520 sequence since mutants YlA (the C-terminus of the propeptide), AIC and Q2R
do not alter the site of cleavage. It is also known that the native structure of the N-terminus in subtilisin does not determine the cleavage site because the 075-variants are processed correctly. Since it is aot yet Imown how to alter the processing site, interactions with the existing N-terminus may be optimized.
Examination of the structure of S 15 subtilisin revealed numerous possibilities for improving stability of the mutant enzyme. The regions of the structure most affected by the deletion are the N-terminal amino acids 1-8, the 36-45 cv-Ioop, the 70-74 a-helix the 84-89 helix turn and the 202-ZI9 B-ribbon.
As previously stated, the first four residues in X75-83 subtilisin are disordered in the. x-ray structure since all its interactions had been with the calcium Ioop.
N-terminal sequencing shows, however, that the first four amino acids are present confirming that processing occurs at the normal site. Other than the N-terminus, there are three other residues whose side chain conformations are distinctly different from wild type. Y6 swings out of a surface niche into a more solvent-exposed position, as an indirect effect of the destabilization of the N-terminus. D4I, a former calcium Iigand, and Y214 undergo a coordinated rearrangement, forming a new hydrogen bond. The B-factors of all three residues increase significantly due to the deletion of amino acids 75-83. In addition, S87 and A88 do not change conformation but exhibit significantly increased B-factors. P86 terminates the a-helix from which the calcium loop was deleted. In view of the above, other mutations at one or more of the above mentioned sites, or at the amino acids proximate thereto, will provide for subtilisin BPN' mutants comprising greater enzymatic activity or increased stability.
There are several logical strategies for remodeling this region of the protein to produce subtilisin BPN' mutants comprising greater enzymatic activity or increased stability. Since the N-terminal four amino acids are disordered in the x-ray structure, one possible approach would be to delete them from the protein. The requirements for processing the propeptide from the mature protein w0 96109396 ~ ~ ~ ~ ~ ~~ ~ PCT/US95105520 are not understood, however. Inserting or deleting amino acids from the N-terminal region is, therefore, problematic. For this reason insertions and deletions in the N-terminal region were avoided in favor of amino acid substitutions. Many of the original amino acids in the above described regions of subtilisin which interacted with the amino acids 75-83 loop can be assumed to no longer be optimal. It was, therefore, possible to increase the stability of the molecule by substituting, deleting or adding at Ieast one amino acid at positions whose environment was changed by the 75-83 deletion.
The first attempt was to mutate the proline at position 5 to alanine to create more flexibility at position 5. This increased flexibility allows the N-terminus to try to find a unique position along the new surface of the protein, created by deletion of the calcium loop. Once the N-terminus assumes a unique location its local interactions may then be optimized.
The PSA mutation was made to try to create more flexibility for the N-terminus and allow it to find a unique position along the new surface of the protein that was created by deletion of the calcium loop. In the native structure, the first five amino acids are in an extended conformation and form B-pair hydrogen bonds with the calcium loop as well as the Q2 side chain interaction with the calcium. The proline at position 5, which is conserved among seven bacterial subtilisins which have a homologous calcium A-site, may help stabilize the extended conformation. The PSA mutation in X75-83 subtilisin should thus result in an increase in the cooperativiry of the unfolding reaction. The X-ray structure of this variant has been determined to 1.8 A.
In toto, the present inventors selected amino acids at ten different positions whose environment had changed substantially for substitution. A
mutagenesis and screening procedure was developed in order to screen all possible substitutions at a particular site. The technique for generating and screening subtilisin variants involves in vitro mutagenesis of the cloned subtiIisin gene, expression of the mutated genes in B. subtilis, and screening for enhanced stability.

' ~ w0 96!09396 2 2 a 0 5 ~ 3 pCT~S95105520 For example, site-directed mutagenesis was performed on the S46 subtiIisin gene using oligormcieotides which were degenerate at one codon. The degeneratc codon contained all combinations of the sequence NNB, where N is any of the four nucleotides and B is T, C or G. The 48 codons represented in this population encode for all twenty amino acids but exclude the ochre and umber termination codons. The mutagenized genes were used to transform B.
subtilis. Examples of particular mutations are shown in Table II as follows:

F
WO 96/09396 2 ~ p p 5 6 3 P~.T/US95105520 U
U
~ C7 ~ t' E
E a CS U
v~~~. e~ a a c~~~~ a~ vv VC~a~ ~~ ~U UC7 UC~,7E"~ ZEa» C~7C~ aCC7.? ~°
a~ZVa z~ ~~ aE~- ~~°-.
jazz ~.a zz z~
~avz ~~ z~ v Uv~'H ~Q ~~ as o E~~~ F'~ ~~ E"a '~ ~ c~Qa~ ~~ ~c~ H~
U~»c~~- ~.~ as 'R' O ~ CU? a C7 R
E ~~ aCU7UU Cd,7U C~7U UU

y U s°~
.. c 'N O
z a~ > ~
ris E ~U ccr~ aw J c ~.» c c rn ~-~ en cn et '~ ~ o a~>~, ax ~a aa.
R

V
C G C
C
O R ~ ~O
.C
O _C ~ 2S O V
O ~~ ~ V'7 fV
~' Cy C; z ° ~ N A

i ~ 2 0 0 ~ 6 3 PGT/U595105520 To have a 98 % chance of fording tryptopban, glutamine, glutamate or methionine in the mutant population, one must screen about 200 mutant clones.
Each of those codons is represented by only one of the 48 codons contained in the population of sequences NNB. Codons for all other amino acids are S represented by at least two codons in the popuIaLion and would require screening of about 100 mutant clones to have a 98 9b chance of being represented in the mutant population.
To identify the optimum amino acid at a position, mutants were screened for retention of enzymatic activity at high temperature. 100 ,uI of media was IO dispensed in each of the 96 wells of a microtiter dish. Each well was inoculated with a Bacillr~s transformant and incubated at 37° with shaking. After 18 hours of growth, 20 ~1 of culture was diluted into 80 ~,1 of 100 mM Tris-HCI, pH 8.0 in a second microtiter dish. This dish was then incubated for one hour at 65°C.
The dish was allowed to cool to room temperature following the high 15 . temperature incubation and 100 ~cl of 1 mM SA.A.PF-pNA was added to each well. The wells which cleaved the pNA (turned yellow) quickest were deemed to contain the most heat resistant subtilisin mutant. Once preliminary identification of a stable mutant was made from the second microliter dish, the Bacillus clone in the corresponding well in the first microliter dish was grown up for further 20 analysis.
The screening procedure identified stabilizing mutations at seven of the ten positions which were examined. As noted, these amino acid positions were selected at positions of the protein whose environment has changed substantially by virtue of the calcium domain deletion. No mutations were identified at 25 positions 4, 74 and 214 which by themselves significantly increased the half Iife of the mutant relative to the parent subtilisin. However, at position 214 the effect of only hydrophobic amino acids was screened. No mutations were found at positions 5, 4I and 43 which resulted in measurable but modest increases in stability. Moreover, several mutations were found at positions at 2, 3, 73, and WO 96!09396 G ~ PCT/US95l05520 206 which signif increased the half mutant relative to candy life of the tile parent subtilisin. These Table III as follows:
stabilizing mutations are shown in Table III - Stabilizing Mutations Region of protein Site Increase N-terminus: Q2K 2.0-foid~

63-85 cx-helix: A73L 2.6-fold 202-220 (3-ribbon Q206V 4.5-fold N-terminus - ~3-ribbonS3C-Q206C (disulfide)14-fold Stabilizing amino acid modifications at positions 2(I~, 73(L) and 2060 were then combined to create subtilisin S73. The properties of S73 subtilisin as well as S46, S79 and S86 are summarized in Table IV.

a Table IV
Mutant Mutations 1 Specific activity2 Half life Increase (60°C)3 S~ - 100U/mg 2.3 min -S73 QZK 160U/mg 25 min 11-fold 3 S79 QZK N.D.4 18 min 8-fold S86 Q2C 85U/mg 80 min 35-fold 1) AlI of subtilisins S4b, 573, S79 and S86 contain the mutations MSOF, Y217K
and N2I8S and Q271E.
2) Specific activity is measured against succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide(SAAPF-pNA) in lOmM Tris-HCI, pH 8.0 a ZS'C.
3) Half life is measured at 60'C in IOmM Tris-HCI, pH 8.0 SOmM NaCI and lOmM
EDTA.
4) Nat determined.
Disulfide bond was formed between the cysteines at positions 3 and 206. The fotTnation of a disulfide band was confirmed by measuring the radius of gyration of the denatured protein by gel electrophoresis.
In many cases, the choice of amino acid at a particular position will be influenced by the amino acids at neighboring positions. Therefore, in order to find the best combinations of stabilizing amino acids, it will be necessary in some cases to vary the amino acids at rivo or more positions simultaneously.
In particular, this was effected at positions 3 and 206 with amino acids whose side chains can potentially interact. It was determined that the best combination of modifications was cysteine modifications at positions 3 and 206. This modification was denoted as S86. Because of the close proximity and suitable geometry between the cysteines at these two positions a disulfide cross-/ink forms ~spontaneousiy between these two residues.
The stability of the S86 subtilisin was studied in relation to S73. It was found that the half Iife of S86 is 80 minutes at 60°C in lOmM Tris-HC1, gH
8.0, SOmM NaC 1 and lOmM EDTA, a 3.2-fold enhancement relative to S73 subtilisin. By contrast, a 3-206 disulfide cross-link would not be able to form in native subtilisins which contain the calcium A-site because the 75-83 binding loop separates the N-terminal amino acids from the 202-219 ~3 ribbon.
Therefore, the enhancement in stability which occurs in the subject S86 mutant lacking the 75-83 binding loop will likely not be observed with native subtilisins similarly cysteine modified at these positions.
It is expected that similar enhancement in stability will be inherent to other subtilisins of the I-S1 and I-S2 group if their calcium loops were deleted (see Siezen et al., Protein Enqineerina, 4, pp. 719-737 at Figure 7). This is a reasonable expectation based on the fact that the primary calcium site in these different subtilisins are formed from almost identical 9 residue loops comprised in the identical position of helix C.
X-ray structures of the I-S1 subtilisins BPN and Carlsberg, as well as the I-S2 subtilisin (savinase), have been determined to high resolution. Comparison of these structures demonstrates that all three have almost identical calcium A-sites.
The x-ray structure of the class I subtilise, thermitase from Thermoactinomyces vulgaris, is also known. Though the overall homology of BPN' to thermitase is much lower than the homology of BPN' to I-S1 and I-S2 subtilisins, thermitases has been shown to have an analogous calcium A-site.
In the case of thermitase, the loop is a ten residue interruption at the identical site in helix C.
Thus, it is expected that the stabilizing mutations exemplified herein will impart similar beneficial effects on stability for the calcium loop-deleted ?5 versions of other class I subtilises.
The stability of S73, S79, and S86 subtilisins relative to S46 subtilisin was compared by measuring their resistance to thermal inactivation at 60°C in 10mM Tris-HCI, pH 8.0, 50mM NaCI and 10mM
EDTA. Aliquots were removed at intervals and the activity remaining in each aliquot was determined. Under these WU 96109396 2 2 4 0 5 ~ ~ p~~S95/05520 conditions, the half life of S4b subtilisin is 2.3 minutes and the half Iife of S73 is 25 minutes (Table IV).
In order to identify other mutants having increased stability any mutagenesis technique known by those skilled in the art may be used. One example of such a technique for generating and screening subtilisin variants involves three steps: 1) in vitro mutagenesis of the cloned subtilisin gene;
2) expression of mutated genes in B. subtilis, and 3) screening for enhanced stability. The key element in the random mutagenesis approach is being able to screen large numbers of variants.
Although random mutagenesis may be employed, the mutagenesis procedure described above allows for mutations to be directed to localized regions of the protein (e.g., the N-terminal region). As noted supra, the S4b, S47, S68, S73, S79 and S86 mutants (which comprise the active-site S221) were found to be enzymatically active. It is expected that other substitutions may be identified which provide for equivalent or even greater stability and activity.
The activities of examples of the calcium-free subtilisin mutants of the present invention against the substrate sAAPF-pNA in Tris-HCI, pH 8.0 and 25°C are given in Table V as follows:
Table V
Subtilisin Specific activity Half Iife (55°C) BPN' 80 U/mg 2 min S12 0.0025 U/mg N.D.1 SIS 0.0025 U/mg N.D.1 S39 0.0025 Ulmg N.D.1 S46 125 U/mg 22 min S47 90 U/mg 4.7 min S68 -100 Ulmg 25 min I) Half lives were nac determined for inacrive subtilisins.

w0 96!09396 Z ~ p p 5 6 3 P~~S95105520 As shown above, the subtilisin mutants S46, S47, 568, S73, S79 and S86 have enhanced catalytic activity in comparison with subtilisin BPN'. Changes in catalytic efficiency due to the deletion were not expected because of the fact that the active site of subtilisin is spatially distant from the calcium A-site.
The stability of these mutant subtilisias was compared to native subtilisin BPN' by measuring their resistance to thermal inactivation. Since the stability of the calcium-free subtilisin mutants should be unaffected by metal chelating agents, the experiment was carried out in lOmM Tris-HCI, pH 8.0, SOmM NaCI
and lOmM EDTA (the association constant of EDTA for calcium is 2 x 10$
M-I). The proteins were dissolved in this buffer and heated to 55°C.
Aliquots were removed at intervals and the activity remaining in each aliquot was determined. The kinetics of inactivation are plotted in Figure 10. Under these conditions, the half life of the subtilisin mutants was much improved over that of subtilisin BPN'. These results indicate that subtilisins which have been mutated IS to eliminate calcium binding at site A have full catalytic activity and improved stability in EDTA relative to subtilisin BPN'. A reasonable level of stability in S46 was achieved even without additional mutations to compensate for Iost interactions resulting from deleting amino acids 75-83.
Thus, the present inventors have provided convincing evidence that subtilisin mutants may be obtained which remain active and yet do not bind calcium. It is expected therefore that these mutants may be utilized in industrial environments that comprise chelating agents. While this has only been specifically shown with subtiIisin BPN', equivalent mutations should work with other serine proteases ~as well, most particularly other I-S 1 or I-S2 subtilisins given that these subtiIisins possess substantial sequence similarity, especially in the calcium binding site.
Such strategies, for example, may involve comparing the sequence of subtilisin BPN' to other serine proteases in order to identify the amino acids which are suspected to be necessary for calcium binding and then making suitable modifications, e.g., by site-specific mutagenesis. Since many subtilisins are WO 96109396 2 2 0 0 ~ fi ~ pCT~S95105520 related to subtilisin BPN' not only through their primary sequences and enzymological properties, but also by X-ray crystallographic data, it is expected that other active subtilisin mutants which lack calcium binding may be produced by site specific mutagenesis. For example, structural evidence exists that the homologous enzyme subtiIisin Carlsberg also comprises two calcium binding sites. Similarly, the X-ray structure of thermitase is known and this subtilisin has an analogous calcium A binding-site to that of subtiIisin BPN'. For thermitase, the calcium binding loop is a ten residue interruption at the identical site in helix C. Accordingly, these enzymes should also be amenable to the mutations described herein which eliminate the calcium binding site and produce a stable, active enzyme. Moreover, as discussed supra, Siezen et aI, has demonstrated that the primary calcium binding site in all subtiIisins in groups I-S I and I-S2 are formed from almost identical nine residue loops in the identical position of helix C. Thus, in view of the almost identical structures of the calcium A-sites, the methods described herein should be applicable to most if not all of the subtilisins in groups I-S1 and I-S2 set forth in Siezen et aI.
Alternatively, if the amino acids which comprise the calcium binding sites are already known for a particular subtilisin, the corresponding DNA will be mutated by site specific mutagenesis to delete one or more of such amino acids, or to provide substitution, deletion or addition mutations which eliminate calcium binding.
The subject mutant subtilisins will generally be produced by recombinant methods, in particular by expression of a subtilisin DNA which has been mutated such that upon expression it results in a subtilisin protein which is enzymatically active and which does not bind calcium.
Preferably, the subtilisin DNA's will be expressed in microbial host cells, in particular Bacillur subtilis, because this bacteria naturally produces subtilisin, is an efficient secretor of proteins, and is able to produce the protein in an active conformation. However, the invention is not restricted to the expression of the subtilisin mutant in Bacillus, but rather embraces expression in any host cell w0 96109396 ~ ~ ~ ~ ~ ~ ~ PCTlUS95105520 which provides for expression of the desired subtiIisin mutants. Suitable host cells for expression are well known in the art and include, e.g., bacterial host cells such as Escherichia coli, Bacillur, Salmonella, Psercdomonas; yeast cells such as Saccharorrryces cerevisiae, Pichia pastoris, Kluveronryces, Candida, Schizosaccharo»ryces; and mammalian host cells such as CHO cells. Bacterial host cells, however, are the preferred host cells for expression.
Expression of the subtilisin DNA will be provided using available vectors and regulatory sequences. The actual selection will depend in Large part upon the particular host cells which are utilized for expression. For example, if the subtilisin mutant DNA is expressed in Bacillus, a Bacillus promoter will generally.be utilized as well as a Bacillus derived vector. The present inventors in particular used the pUBlIO-based expression vector and the native promoter from the subtilisin BPN' gene to control expression on Bacillus subtilis.
It is further noted that once the amino acid sequence of a particular subtilisin mutant which does not bind calcium has been elucidated, it may also be possible to make the subtiiisin mutant by protein synthesis, e.g., by Merrifield synthesis. However, expression of the subtilisin mutants in microbial host cells will generally be preferred since this will allow for the microbial host cell to produce the subtilisin protein in a proper conformation for enzymatic activity.
However, since the present inventors further teach herein a method for obtaining in virro refolding of the subtilisin mutant, it should be possible to convert improperly folded subtilisin mutants into an active conformation.
In order to further illustrate the present invention and the advantages thereof, the following specific examples are given, it being understood that the same is intended only as illustrative and in nowise limitative.

FX~MPI FS
Example 1 Cloning and Expression The subtilisin gene from Bacillus amyloliquefaciens (subtilisin BPN') has been cloned, sequenced, and expressed at high levels from its natural promoter sequences in Bacillus subtilis (Wells et al., Nucleic Acids Res. 11:7911-7925 (1983); Vasantha et al., J. Bacteriol. 159:811-819 (1984)). All mutant genes were recloned into a pUB110-based expression plasmid and used to transform 8. subtilis. The B.
subtilis strain used as the host contains a chromosomal deletion of its subtilisin gene and therefore produces no background wild type (wt) activity (Fahnestock et al., Appl. Environ. Microbial. 53:379-384 (1987)).
Oligonucleotide mutagenesis was carried out as previously described. (Zoller et al., Methods Enzymol. 100:468-500 (1983); Bryan et al., Proc. Natl. Acad.
Sci. 83:3743-3745 (1986b)). S221 C was expressed in a 1.51 New Brunswick I 5 fermentor at a level of approximately 100 mg of the correctly processed mature form per liter. The addition of wild type subtilisin to promote production of the mature form of S221 C subtilisin was not required in our bacillus host strain as was the case for prior strains (Abrahmsen et al., Biochemistry 30:4151-4159 (1991 )).
Protein Purification & Characterization. Wild type subtilisin and the variant enzymes were purified and verified for homogeneity essentially as described in Bryan et al., Proc. Natl. Acad. Sci. 83:3743-3745 (1986b);
Pantoliano et al., Biochemistry 27:8311-8317 (1988). In some cases the C221 mutant subtilisins were re-purified on a sulfhydryl specific mercury affinity column (Affi-gel~ 501, Biorad). Assays of peptidase activity were performed by monitoring the hydrolysis of succinyl-(L)-Ala-(L)-Ala-(L)-Pro-(L)-Phe-p-nitroanilide, hereinafter sAAPFna, as described by DeIMar et al., Anal Biochem. 99:316-320 (1979). The protein concentration, [P], was determined using P°''°~° = 1.17 at 280 nm (Pantoliano et al., Biochemistry 28:7205-7213 (1989)). For variants which contain the Y217K change, the P°v°~° at 280nm was calculated to be 1.15 (or 0.96 X wt), based on WO 96!09396 2 2 0 0 ~ 6 3 the loss of one Tyr residue (Pantoliano et al., Biochemistry 28:7205-7213 (1989)).
N terminal Analysis The first five amino acids of subtilisin S 15 were determined by sequential Edman degradation and ~HPLC analysis. This revealed S that 100% of the material had the amino acid sequence expected from the DNA
sequence of the gene and that processing of the pro-peptide was at the same position in the sequence for the mutant as for the wild type enzyme.
xam Ie 2 Structure of the calcium A site of S12 subtilisin Calcium at site A is coordinated by five carbonyl oxygen ligands and one aspartic acid. Four of the carbonyl oxygen ligands to the calcium are provided by a loop composed of amino acids 75-83 (Figure 2). The geometry of the ligands is that of a pentagonal bipyramid whose axis runs through the carbonyls of 75 and 79. On one side of the loop is the bidentate carboxylate (D41), while on the other side is the N-terminus of the protein and the side chain of Q2. The seven coordination distances range from 2.3 to 2.6 A, the shortest being to the asparryl carboxylate.
Three hydrogen bonds link the N-terminal segment to loop residues 78-82 in parallel-beta arrangement.
Preparation of apo-subtilisin S 11 and S I2 subtilisin contain an equal molar amount of tightly bound calcium after purification. X-ray crystallography has shown this calcium to be bound to the A site (Finzel et al., J. Cell.
Biochem.
a 1. 10A:272 (1986); Pantoliano et al., Biochemistry 27:8311-8317 (1988);
McPhalen et al., Biochemistry 27:6582-6598 (1988)).
Complete removal of calcium from subtilisin is very slow, requiring 24 hours of dialysis against FrDTA at 25°C to remove all calcium from the protein and then 48 more hours of dialysis in high salt (Brown et al., Bivchemistrv 16:3883-3896 (1977)) at 4°C to remove all FrDTA from the protcin. To prepare the calcium-free form of subtilisins S 11 and S 12, 20mg of lyophilized protein was dissolved in SmI of lOmM FrDTA, IOmM tris(hydrvxymethyl)amino-methane hydrochloric acid (hereinafter Tris-HCI) at pH 7.5 and dialyzed against the same buffer for 24 hours at 25°C. In order to remove EDTA, which binds to subtilisin at low ionic strength, the protein was then dialyzed twice against liters of 0.9M NaCI, 10mM Tris-HCI at pH 7.5 at 4°C for a total of 24 hours and then three times against 2 liters of 2.5mM Tris-HCI at pH 7.5 at 4°C
for a total of 24 hours. Chelex° 100 was added to all buffers not containing EDTA.
When versions of C221 subtilisin not containing stabilizing amino acid substitutions were used, up to 50% of the protein precipitated during this procedure. It is essential to use pure native apoenzyme in titration experiments so that spurious heat produced by precipitation upon the addition of calcium does not interfere with the measurement of the heat of binding.
To ensure that preparations of apo-subtilisin were not contaminated with calcium or EDTA, samples were checked by titration with calcium in the presence of Quin2 prior to performing titration calorimetry.
1 ~ Titration Calorimetry Measurements The calorimetric titrations were performed with a Microcal Omega titration calorimeter as described in detail by Wiseman et al., Analytical Biochemistry 179:131-137 (1989). The titration calorimeter consists of a matched reference cell containing the buffer and a solution cell (1.374 mL) containing the protein solution. Microliter aliquots of the ligand solution are added to the solution cell through a rotating stirrer syringe operated with a plunger driven by a stepping motor. After a stable baseline was achieved at a given temperature, the automated injections were initiated and the accompanying heat change per injection was determined by a thermocouple sensor between the cells. During each injection, a sharp exothermic peak appeared which returned to the baseline prior to the next injection occurring 4 minutes later. The area of each peak represents the amount of heat accompanying binding of the added ligand to the protein. The total heat (Q) was then fit by a nonlinear least squares minimization method (Wiseman et al., Analytical Biochemistry 179:131-137 (1989)) to the total ligand concentration, [Ca]total, according to the equation:

WO 96109396 2 2 0 0 5 ~ 3 p~/Ug95105520 dQ/d[Ca]toy = DH[1~2+(1-(1+r)/2-Xr/2)/Xr-2Xr(1-r)+1+r2)1~2] (1) wherein 1/r = (P]to~xKa and Xr = [Ca]to~/[Plto~.
The binding of calcium to the S11 and S12 subtilisins was measured by titration calorimetry as it allows both the binding constant and the enthalpy of .
binding to be determined (Wiseman et aL, Analytical Biochemistry 179:-131-137 (1989); Schwarz et al., J. Bioi. Chem. 266:24344-24350 (1991)).
The S 11 and S I2 subtilisin mutants were used in titration experiments because production of the wild type apoenzyme is impossible due to its proteolytic activity and Iow stability. Titrations of S 11 and S 12 were performed at protein concentrations [P] = 30~,M and IOO~cM. Titration of the S I 1 apoenzyme with calcium at 25°C is shown in Figure 4. The data points correspond to the negative heat of calcium bindiag associated with each titration of calcium. The titration calorimeter is sensitive to changes in Ka under conditions at which the product of Ka x [P] is between 1 and 1000 (Wiseman et . al., Analytical Biochemistry 179:131-137 (1989)). Since the Ka for subtilisin is about 1x107 M-1, these protein concentrations result in values of Ka x [P] =

and 1000. At lower protein concentrations the amount of heat produced per titration is difficult to measure accurately.
_ The results of fitting the titrations of S I 1 and S 12 to a calculated curve ZO are summarized in Table 2. The parameters in the table include binding parameters for stoichiometric ratio (n), biading constant (Ka) and binding enthalpy (D~. These parameters were determined from deconvolution using nonlinear least squares minimization (Wiseman et al., Analytical Biochemistry 179:131-137 (1989)). Measurements for each experimental condition were performed in duplicate at 25°C. The protein concentrations ranged from 30 to 100 ~,M while the concentration of the calcium solutions were about 20 times the protein concentrations. Each binding constant and enthalpy were based on several titration runs at different concentrations. Titration tuns were performed until the titration peaks were close to the baseline.

' Z2 00 563 w0 96/09396 PCT/US95105520 TABLE 2: Titration Calorimetry of the Calcium A Site in Subtilisin Mutants Sll and SI2.
Mutant (P] Parameters calculated from fit n ~ pH
S I 1 100~cM 0. 98 0.01 7. 8 0.2 x -11.3 0.

SI1 33~cM 0.90.3 6.8/.5x106 -10.90.2 S12 IOO~,cM 0.99 0.01 6.4 0.2 x 106 -11.8 0.5 The average values obtained are similar for S 11 and S I2: DH = - -1 I
kcal/mol; Ka = 7 x 106 M-I and a stoichiometry of binding of 1 calcium site per molecule. The weak binding site B does not bind calcium at concentrations below the millimolar range, and therefore does not interfere with measurement of binding to the binding site A. The standard free energy of binding at 25°C is 9.3 kcallmol. The binding of calcium is therefore primarily enthalpically driven with only a small net Loss in entropy (~Sb~g = -6.7 cal/°moI).
am Ie 3 In vitro refolding of SIS subtilisin. For refolding studies subtilisin was maintained as a stock solution in 2.5 mM Tris-HCI at pH 7.5 and SOmM KCl at a concentration of approximately 100~.M. The protein was denatured by diluting the stock solution into SM guanidine hydrochloride (Gu-HCI) at pH 7.5 or in most cases into 25 mM H3P04 or HCI at pH 1.8 - 2Ø The final concentration of protein was 0.1 to 5 ~cM. S15 was completely denatured in less than 30 seconds by these conditions. S I2 required approximately 60 minutes to became fully denatured. Acid-denatured protein was then neutralized to pH 7.5 by the addition of Tris-base (if denatured in HCl) or SM NaOH (if denatured in H3P0~. Refolding was initiated by the addition of KCl, NaC1 or CaCl2 to the WO 96109396 . ~ 3 PCT/US95/05520 desired concentration. Fvr example, KCl was added from a stock solution of 4M
to a final concentration of 0.1 to I.SM with rapid stirring. In most cases renaturation was carried out at 25°C. The rate of renaturation was determined spectrophotometrically by uv absorption from the increase in extinction at ~ _ 286, from the increase in intrinsic tyrosine and tryptophan fluorescence (excitation ~ = 282, emission ~ - 347), or by circular dichroism from the increase in negative ellipticity at A = 222 nm.
xam to 4 X ray Crystallography. Large single crystal growth and X-ray diffraction data collection were performed essentially as previously reported (Bryan et a.l., Proteins: Struct. Funct. Genet. 1:326-334 (1986a); Pantoliano et al., Biochemistry 27:8311-8317 (1988); Paatoliano et aL, Biochemistry 28:7205-7213 (1989)) except that it was not necessary to inactivate the S221C variants with diisopropyl fluorophosphate (DFP) in order to obtain suitable crystals. The starting model for S I2 was made from the hyperstable subtiIisin mutant 8350 (Protein Data Bank entry lSOl.pdb). The SIZ structure was refined and then modified to provide the starting model for S 15.
Data sets with about 20,000 reflections between 8.0 A and 1.8 A
resolution were used'to refine both models using restrained least-squares techniques (Hendrickson et al., "Computing in Crystallography" in Diamond et aL, eds., Bansalore: Indian Institute of Science 13.01-13.23 (1980)). Initial difference maps for S I5, phased by a version of S 12 with the entire site A
region omitted, clearly showed continuous density representing the uninterrupted helix, permitting an initial S 15 model to be constructed and refinement begun. Each mutant was refined from R approximately 0.30 to R approximately 0.18 in about eighty cycles, interspersed with calculations of electron density maps and manual adjustments using the graphics modeling program FRODO (Jones, J. Appl.
Crystallogr. 11:268-272 (1978)).

WO 96/09396 2 2 ~ 0 5 6 3 pCTlLTS95105520 Except for the region of the deleted calcium-binding loop, the structures of S12 and S15 are very similar, with a root mean square (r.m.s) deviation of 0.18 A between 262 a-carbons. The N-terminus of S I2 (as in the wild-type) lies beside the site A loop, furnishing one calcium coordination ligand, the side chain oxygen of Q2. In S IS the loop is gone, leaving residues I-4 disordered. In S

(as in wild type) the site A loop occurs as an interruption in the last turn of a I4-residue alpha helix; in S IS this helix is uninterrupted and shows normal helical geometry over its entire length. Diffuse difference density and higher temperature factors indicate some disorder in the newly exposed residues adjacent IO to the deletion.
x le 5 Di,~erenrial Scanning Calorimerry The stability properties of S 12 and S IS
were studied using DSC (differential scanning calorimetry). The X75-83 mutant (S IS) is very similar in melting temperature to the apoenzyme of S 12. The DSC
profiles of apo-S I2 and S IS are shown in Figure 3. The temperature of maximum heat capacity is 63.0°C for SIS and 63.5°C for apo-S12 at pH 9.63.
The DSC experiments were carried out at high pH to avoid aggregation during the denaturation process. The amount of excess heat absorbed by a protein sample as the temperature increased through a transition from the folded to unfolded, state at constant pressure, which provided a direct measurement of the ~Fi of unfolding (Privalov et aL, Methods Enzvmol. I3I:4-51 (1986)). ~Tc~ of unfolding for apo-S 12 and S 15 is about 140 kcal/mol. Above gH 10.0, the unfolding transition for S 15 fit a two-state model reasonably well, consistent with equilibrium thermodynamics as expressed in the van't Hoff equation (dln K / dT
= ~H I (RT2)) with OHS,g (the van't Hoff enthalpy or apparent enthalpy) approximately equal to ~H~ (the calorimetric or true enthalpy). At pH 9.63, however, the melting profile for both proteins was asymmetric indicating that the unfolding is not a pure two-state process.

' ~ w0 96109396 ~ 2 p 0 ~ 6 3 PCTlUS95105320 c le 6 Measuring kinetics of calcium dissociation. The dISSOCIaIIOn of calcium from subtilisin is a slow process. To measure this rate the fluorescent calcium chelator Quire 2 was used. Quire 2 binds calcium with a Ka of 1.8 z 108 at pH
7.5 (Linse et al., Biochemistry 26:6723-5735 (1987)). The fluorescence of Quire 2 at 495nm increases by approximately 6-fold when bound to calcium (Bryant, Biochem. J. 226:613-616 (1985)). Subtilisin SI1 or S12 as isolated contains one calcium ion per molecule. When mixed with an excess of Quip 2, the kinetics of calcium release from the protein can be followed from the increase in fluorescence at 495nm. The reaction is assumed to follow the pathway N(Ca) ~
N + Ca + Quire 2 a Quin(Ca). The dissociation of calcium from subtilisin is very slow relative to calcium binding by Quire 2, such that the change in fluorescence of Quire 2 is equal to the rate of calcium dissociation from subtilisin.
As can be seen in Figure Sa, the initial release of calcium from S I I follows simple first order kinetics.
Temperature dependence of calcium dissociation The first order rate constant (k) for calcium dissociation was measured from 20 ° to 45 °C. The plot of In k vs. 1/T°K is roughly linear. The calcium dissociation data was curve fit using transition state theory according to the Erying equation:
~G$=-RT In KT=-RT In kh/kgT (2) wherein kg is the Boltmaan constant, h is Planck's constant and k is the first order rate constant for folding. A graph of In hklkgT vs. 1lT is shown in Figure Sb.
The data was then curve fit according to the equation (Chen et al. , Biochemistry 28:691-599 (1989)):
In KT = A + B(To/T) + C In (ToIT) (3) wherein A = [OCpT + GST(To)]/R; B = A - OG#(T~/RTo; C = ~Cp~IR. The data obtained yields the following results: OG~ = 22.7 kcal/mol; ~Cp'~ _ -0.2 kcal/°mol; OS'+ _ -IO call°mol; and ~H'~ = 19.7 kcallmol at a reference temperature of 25°C. A possible slight curvature of the plot would be due to a change in heat capacity associated with formation of the transition state (~Cp'$ = 0.2 kcal/°mol). ~Cp for protein folding has been shown to be closely correlated with a change in exposure of hydrophobic groups to water (Privalov et al., adv. Protein Chem. 39:191-234 (1988); Livingstone et al., Biochemistry 30:4237-4244 (1991 )). In terms of heat capacity, the transition state therefore appears similar to the native protein. The values for ~S'$ and OH'$ obtained from Figure 5b indicate that the transition state is enthalpically less favorable than the calcium bound form with only a small change in entropy.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

22 00 ~s~
WO 96109396 CTIUS95l05520 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: BRYAN, Philip N
ALEXANDER, Patrick STRAUSBERG, Susan L
(ii) TITLE OF INVENTION: CALCIUM FREE SUBTILISIN MUTANTS
(iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Burns, Doane, Swecker & Mathis (8) STREET: P.O. Box 1404 (C) CITY: Alexandria (D) STATE: Virginia (Ej COUNTRY: United States (F) ZIP: 22313-1404 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #~1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Meuth, Donna M
(B) REGISTRATION NUMBER: 36,607 (C) REFER.ENCE/DOCRET NUMBER: 028755-021 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (703) 836-6620 (B) TELEFAX: (703) 836-2021 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 1868 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE:
(A) NAME/KEY: CDS
(Bj LOCATION: 450..1599 (ix) FEATURE:
(A) NAME/KEY: mat peptide (B) LOCATION: 772..1599 (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 450 (D) OTHER INFORMATION: /note= "Amino Acid Val at position 450 iS fMet."

' 1 WO 96/09396 PGTlUS95/05520 -41- 22 p0 X63 (xi) SEQUENCE SEQ ID NO:1:
DESCRIPTION:

TGACAATATC
AACATCAATG
ATATTCATTA

TTTCTCGGTC
AAGAAAGCCA
AAGACTGATT

CAACAAAAGA
TGACATTTAT
CCTGTTTTTG

TCGACCCAGG
AAACAAGCGA
GTGATTGCTC

GTTTTTTCCA
TTAAAATTTA
AATATTTCGA

CCTAAATAGA
AATAAAACAA
TCTGAAAAAA

ATACAATTAA
TACACAGAAT
AATCTGTCTA

GGATAAAGA
GTG
AGA
GGC
AAA
AAA
GTA

Val Arg Gly Lys Lys Trp Ile Val CTG GCT TTA TTC

Ser Leu Phe Leu Ala Ile Phe Thr Met Ala Gly Ser Leu Ala Leu Phe TCT CAG GGG AAG

Thr Ser Ala Ala Ala Lys Ser Asn Gly Glu Lys Tyr Ser Gln Gly Lys GGG AAA ATG GCT

Ile Va1 Phe Gln Thr Ser Thr Met Ser Ala Lys Lys G1y Lys Met Ala GTC TCT GGC CAA

Lys Asp Ile Glu Lys Gly Lys Val G1n Lys Phe Lys Val Ser Gly Gln TAT GTA~GACGCA TCA GCT TTA AAC GAA AAA GCT AAA GAA 713 GCT ACA GTA

Tyr Val Ala Ser Ala Leu Asn G1u Lys Ala Lys Glu Asp Ala Thr Val -35 ~ -30 -25 -20 AAA CCG GCT CAC

Leu Lys Asp Ser Val Tyr Val Glu Glu Asp Val Ala Lys Pro Ala His TAC CAG CCT ATT

His Ala Ala Ser Val Tyr Gly Val Ser Gln Lys Ala Tyr Gln Pro Ile CTG TCT TAC AAA

Pro Ala His Gln Gly Thr Gly Ser Asn Val Val Ala Leu Ser Tyr Lys GAC GGT TCT AAG

Val Ile Ser Ile Asp Ser His Pro Asp Leu Val Ala Asp Gly Ser Lys GCC ATG TCT CAA

Gly Gly Ser Val Pro Glu Thr Asn Pro Phe Asp Asn Ala Met Ser Gln CAC ACT GCC CTT

Asn Ser Gly His Val Gly Thr Val Ala Ala Asn Asn His Thr Ala Leu GGT TTA GCG TAC

Ser Ile Val Gly Val Pro Ser Ala Ser Leu Gly Leu Ala Tyr Ala Val WO 96!09396 pCT/US95/05520 GAC GGC ATC

Lya Val Leu Gly Ala Asp GlySer GlnTyr Ser Trp IleAsn Gly Ile AAC

Gly Ile G1u Trp AlaIle AlaAsn AsnMetAsp Val Ile MetSer Asn GTT

Leu Gly Gly Pro SerGly SerAla AlaLeuLys Ala Ala AspLys Va1 AAC

Ala Val Ala Ser GlyVal ValVal ValAlaAla A1a Gly GluGly Asn TAC

Thr Ser Gly Ser SerSer ThrVal GlyTyrPro Gly Lys ProSer Tyr GCA

Val Ile Ala Val GlyAla ValAsp SerSerAsn G1n Arg SerPhe Ala GTA

Ser Ser Val Gly ProGlu LeuAsp ValMetAla Pro Gly SerIle Val GGT

Gln Ser Thr Leu ProGly AsnLys TyrGlyAla Tyr Asn ThrSer Gly CTT

Met Ala Ser Pro HisVal AlaGly AlaAlaAla Leu Ile SerLys Leu GAA

His Pro Asn Trp ThrAsn ThrGln ValArgSer Ser Leu AsnThr Glu CTG

Thr Thr Lys Leu GlyAsp SerPhe TyrTyrGly Lys Gly IleAsn Leu GTA CAG GCG GCA GCTCAG TAA CCGGCCTTGG GT 1fi29 A CCCCGCCG
ACATAAAAAA

Val Gln A1a Ala AlaGln CCGCATGTTC ATAATCGACG
GATGGCTCCC

ACGGCGGGTT AGTCCCGTAA
CGGCCAAGTC

CTTCCCGGTT TCAATGCCGT
AACGGTCGGC

AGACGGCATT TCAGAAGCAA
AACTGAGCA

Claims (33)

WE CLAIM:

1. An enzymatically active subtilisin protein which has been mutated to delete amino acids at positions 75-83 of mature subtilisin BPN' to eliminate the ability of said subtilisin protein to bind calcium at a high affinity calcium binding site, wherein the mutated subtilisin protein further includes at least one additional deletion, substitution or addition mutation which enhances the stability of the mutant protein in at least one region which corresponds to a region of the mature subtilisin BPN' selected from the group of mature subtilisin BPN' regions consisting of the N-terminal acids 1-8, the .omega.-loop amino acids 36-45 and the .alpha.-helix amino acids 70-74.

2. The subtilisin protein of claim 1, wherein the mutated subtilisin protein further comprises at least one additional mutation in the amino acid sequence that is outside of the mature subtilisin BPN' regions: N-terminal amino acids 8, the .omega.-loop amino acids 36-45 and the .alpha.-helix amino acids 70-74.

3. The subtilisin protein of claim 1, wherein the mutated subtilisin protein comprises at least one deletion, substitution or addition mutation in the amino acid position or positions selected from the group consisting of: 2, 3, 5, 41, 43, and 73.

4. The subtilisin protein of claim 1, wherein the mutated subtilisin protein further comprises at least one additional mutation in the .beta.-ribbon amino acids 202-219.

5. The subtilisin protein of claim 1 wherein the mutated protein comprises at least one substitution mutation selected from the group consisting of P5S, D41A, K43R, K43N, Q2K, Q2W, Q2L, A73L, A73Q and S3C.

6. The subtilisin protein of claim 5, further comprising at least one substitution mutation selected from the group consisting of N218S, M50F, Y217K, Q271 E, Q206C, Q206V, Q2061 and Q206W.

7. The subtilisin protein of claim 6, wherein the subtilisin mutant comprises the substitution mutations of N218S, M50F, Y217K and P5S.

8. The subtilisin protein of claim 6, wherein the subtilisin mutant comprises the substitution mutations of N218S, M50F, Y217K, Q271 E, Q2K, A73L and Q206V.

9. The subtilisin protein of claim 6, wherein the subtilisin mutant comprises the substitution mutations of N218S, M50F, Y217K, Q271 E, Q2K, A73L and Q206C.

10. The subtilisin protein of claim 1, wherein the subtilisin protein comprises the substitution mutation PSA.

11. The subtilisin protein of claim 10, further comprising at least one substitution mutation selected from the group consisting of N218S, M50F and Y217K.

12. The subtilisin protein of any one of claims 1 to 11, wherein the subtilisin is from a Bacillus strain.

13. The subtilisin protein of any one of claims 1 to 12, wherein the subtilisin protein is selected from the group consisting of a subtilisin BPN' mutant, a subtilisin Carlsberg mutant, a subtilisin DY mutant, a subtilisin amylosacchariticus mutant, a subtilisin mesenticopeptidase mutant and a subtilisin Savinase mutant.

14. The subtilisin protein of claim 13, wherein the subtilisin protein is a subtilisin BPN' mutant.

15. A recombinant method which provides for the expression of an enzymatically active subtilisin protein which has been mutated to delete amino acids at positions 75-83 of mature subtilisin BPN' to eliminate the ability of said subtilisin protein to bind calcium at a high affinity calcium binding site, wherein the mutated subtilisin protein further comprises at least one deletion, substitution or addition mutation which enhances the stability of the mutant protein in at least one region which corresponds to a region of the mature subtilisin BPN' selected from the group of mature subtilisin BPN' regions consisting of the N-terminal amino acids 1-8, the .omega.-loop amino acids 36-and the .alpha.-helix amino acids 70-74, said method comprising:
(a) transforming a recombinant host cell with an expression vector which contains a DNA which encodes said mutated enzymatically active subtilisin protein;
(b) culturing said host cell under conditions which provide the expression of the mutated enzymatically active subtilisin; and (c) recovering the expressed mutated enzymatically active subtilisin from said microbial host.

16. The recombinant method of claim 15, wherein the subtilisin protein further comprises at least one additional mutation in the amino acid sequence that is outside of the mature subtilisin BPN' regions: N-terminal amino acids 8, the .omega.-loop amino acids 36-45 and the .alpha.-helix amino acids 70-74.

17. The recombinant method of claim 15, wherein the mutated subtilisin protein comprises at least one deletion, substitution or addition mutation in the amino acid position or positions selected from the group consisting of: 2, 3, 5, 41, 43, and 73.

18. The recombinant method of claim 15, wherein the mutated subtilisin protein further comprises at least one additional mutation in the .beta.3-ribbon amino acids 202-219

19. The recombinant method of claim 15, wherein the subtilisin protein comprises at least one substitution mutation selected from the group of P5S, D41 A, K43R, K43N, Q2K, Q2W, Q2L, A73L, A73Q, and S3C.

20. The recombinant method of claim 19, further comprising at least one substitution mutation selected from the group consisting of N218S, M50F, Y217K, Q271E, Q206C, Q206V, Q2061 and Q206W.

21. The recombinant method of claim 20, wherein the subtilisin protein comprises the substitution mutations N218S, M50F, Y217K and P5S.

22. The recombinant method of claim 20, wherein the subtilisin protein comprises the substitution mutations N218S, M50F, Y217K, Q271 E, Q2K, A73L and Q206V.

23. A recombinant DNA which encodes a subtilisin protein which has been mutated to delete amino acids at positions 75-83 of mature subtilisin BPN' to eliminate the ability of said subtilisin protein to bind calcium at a high affinity calcium binding site, wherein the mutated subtilisin protein further comprises one or more deletion, substitution or addition mutation which enhances the stability of the mutant protein in at least one region which corresponds to a region of the mature subtilisin BPN' selected from the group of mature subtilisin BPN' regions consisting of the N-terminal amino acids 1-8, the .omega.-loop amino acids 36-45 and the .alpha.-helix amino acids 70-74 and which mutated subtilisin protein retains enzymatic activity and stability.

24. The recombinant DNA of claim 23, wherein the mutated subtilisin protein encoded by said DNA further comprises at least one additional mutation in the amino acid sequence that is outside of the mature subtilisin BPN' regions including: N-terminal amino acids 1-8, the .omega.-loop amino acids 36-45 and the .alpha.-helix amino acids 70-74.

25. The recombinant DNA of claim 24, wherein the mutated subtilisin protein encoded by said DNA comprises at least one deletion, substitution or addition mutation in an amino acid position or positions selected from the group consisting of amino acids: 2, 3, 5, 41, 43, and 73.

26. The recombinant DNA of claim 24, wherein the mutated subtilisin protein encoded by said DNA further comprises at least one additional mutation in the .beta.-ribbon amino acids 202-219.

27. The recombinant DNA of claim 24, which encodes at least one substitution mutation selected from the group consisting of P5S, D41A, K43R, K43N, Q2K, Q2W, Q2L, Q73L, A73Q and S3C.

28. The recombinant DNA of claim 27, further comprising at least one substitution mutation selected from the group consisting of N218S, M50F, Y217K, Q271E, Q206C, Q206V, Q206I and Q206W.

29. The enzymatically active subtilisin of claim 1, which has enhanced thermal stability relative to a mutated protein containing only the mutation eliminating calcium binding, comprising at least one substitution mutation in an amino acid position or positions selected from the group consisting of amino acids: 2, 3, 5, 41, 43 and 73.

30. The enzymatically active subtilisin of claim 29, further comprising at least one substitution mutation in an amino acid position or positions selected from the group consisting of amino acids: 50, 206, 217, 218 and 271.

31. The subtilisin protein of claim 1, wherein the mutated subtilisin further comprises an additional mutation in the helix turn amino acids 84-89.

32. An enzymatically active subtilisin protein which has been mutated to delete amino acids at positions 75-83 of mature subtilisin BPN' to eliminate the ability of said subtilisin protein to bind calcium at a high affinity calcium binding site, wherein the mutated subtilisin protein includes at least one additional deletion, substitution or addition mutation which enhances the stability of the mutant protein in at least one region which corresponds to a region of the mature subtilisin BPN' selected from the group of mature subtilisin BPN' regions consisting of the N-terminal acids 1-8, the .omega.-loop amino acids 36-45, the .alpha.-helix amino acids 70-74 and the helix turn amino acids 84-89, and wherein a mutation in the N-terminal amino acids 1-8, the .omega.-loop amino acids 36-45, the .alpha.-helix amino acids 70-74 and the helix turn amino acids 84-89 is free of disulfide cross-links.

33. A recombinant method which provides for the expression of an enzymatically active subtilisin protein which has been mutated to delete amino acids at positions 75-83 of mature subtilisin BPN' to eliminate the ability of said subtilisin protein to bind calcium at a high affinity calcium binding site, wherein the mutated subtilisin protein and includes at least one deletion, substitution or addition mutation which enhances the stability of the mutant protein in at least one region which corresponds to a region of the mature subtilisin BPN' selected from the group of mature subtilisin BPN' regions consisting of the N-terminal amino acids 1-8, the .omega.-loop amino acids 36-45, the .alpha.-helix amino acids 70-74 and the helix turn amino acids 84-89, and wherein a mutation in the N-terminal amino acids 1-8, the .omega.-loop amino acids 36-45, the .alpha.-helix amino acids 70-74 and the helix turn amino acids 84-89 is free of disulfide cross-links, said method comprising:
(a) transforming a recombinant host cell with an expression vector which contains a DNA which encodes said mutated enzymatically active subtilisin protein;

(b) culturing said host cell under conditions which provide the expression of the mutated enzymatically active subtilisin; and (c) recovering the expressed mutated enzymatically active subtilisin from said microbial host.